Biasiotta et al. J Transl Med (2016) 14:285 DOI 10.1186/s12967-016-1038-y Journal of Translational Medicine

RESEARCH Open Access Ion channels expression and function are strongly modified in solid tumors and vascular malformations Antonella Biasiotta1†, Daniela D’Arcangelo2†, Francesca Passarelli2, Ezio Maria Nicodemi2* and Antonio Facchiano2*

Abstract Background: Several cellular functions relate to ion-channels activity. Physiologically relevant chains of events lead- ing to angiogenesis, cell cycle and different forms of cell death, require transmembrane voltage control. We hypothe- sized that the unordered angiogenesis occurring in solid cancers and vascular malformations might associate, at least in part, to ion-transport alteration. Methods: The expression level of several ion-channels was analyzed in human solid tumor biopsies. Expression of 90 coding for ion-channels related was investigated within the Oncomine database, in 25 independ- ent patients-datasets referring to five histologically-different solid tumors (namely, bladder cancer, glioblastoma, , breast invasive-ductal cancer, carcinoma), in a total of 3673 patients (674 control-samples and 2999 cancer-samples). Furthermore, the ion-channel activity was directly assessed by measuring in vivo the electrical sym- pathetic skin responses (SSR) on the skin of 14 patients affected by the flat port-wine stains vascular malformation, i.e., a non-tumor vascular malformation clinical model. Results: Several ion-channels showed significantly increased expression in tumors (p < 0.0005); nine genes (namely, CACNA1D, FXYD3, FXYD5, HTR3A, KCNE3, KCNE4, KCNN4, CLIC1, TRPM3) showed such significant modification in at least half of datasets investigated for each cancer type. Moreover, in vivo analyses in flat port-wine stains patients showed a significantly reduced SSR in the affected skin as compared to the contralateral healthy skin (p < 0.05), in both latency and amplitude measurements. Conclusions: All together these data identify ion-channel genes showing significantly modified expression in different tumors and cancer-vessels, and indicate a relevant electrophysiological alteration in human vascular mal- formations. Such data suggest a possible role and a potential diagnostic application of the ion–electron transport in vascular disorders underlying tumor neo-angiogenesis and vascular malformations. Keywords: Cancer, Ion-channels, Autonomic nervous system, Sympathetic skin response, SSR, Flat port-wine stains

Background transmembrane voltage represents a fundamental pro- Several key cellular functions are related to transmem- cess in many physiologically relevant steps, includ- brane potentials and lie under the control of ion chan- ing cell cycle progression [1] and different forms of cell nels, pumps and complexes. Controlling death [2, 3]. Over expression or increased activity of ion channels has been demonstrated as a response to mito- gens exposure [4–6]. Several studies show a direct link *Correspondence: [email protected]; [email protected] †Antonella Biasiotta and Daniela D’Arcangelo contributed equally to this between the transmembrane ion flow and carcinogenesis work [7, 8]. However, as pointed out [9], the role membrane 2 Istituto Dermopatico dell’Immacolata, IDI-IRCCS, Fondazione Luigi Maria potential plays in the pathogenesis of several disorders, Monti, via Monti di Creta 104, 00167 Rome, Italy Full list of author information is available at the end of the article including cancer, is still not well understood. Plasma

© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Biasiotta et al. J Transl Med (2016) 14:285 Page 2 of 15

membrane de-polarization has a pivotal role at different we thus hypothesized that ion-transport may represent stages of cell cycle progression and in various cell types a measurable sign of the altered underlying angiogenesis. [9]. Namely, endothelial cells hyper-polarization has We, therefore, measured in vivo the ion-channel func- been shown to contribute to cell division arrest [10], and tion in a human vascular malformation model, namely channels are known to control migratory cellular prop- flat port-wine stains, as a model to test electrical-stimuli erties in wound healing [11]. Further, Ca2+, K+ and Cl− transport in a human vascular disorder accepted by the channels are essential regulators of cell proliferation and Ethic Committee. cancer development [12–16]. As recently further dem- onstrated, several ion-channels are directly involved in Methods controlling tumor—[17] as well as non-tumor angiogen- Ion‑channel expression investigation esis [18]. Expression and activity of TRPV4 channel have levels were investigated by accessing been found suppressed in tumor [19], and human cancer datasets available at Oncomine (http:// its activation has been found to normalize tumor vessels www.oncomine.org). The current Oncomine version [20]; inhibiting Cl− channel activity has anti-angiogenesis contains several hundred different patients-datasets, and anti-glioma properties [21]; finally, human voltage- referring to tumors biopsies obtained from almost any dependent K+ channel has been found associated with histological source; unfortunately vascular tumors (such cancer aggressiveness and angiogenesis [22]. Therefore, as hemangioma, angiomas, hemangioendothelioma, ion-channels play a fundamental role in cancer progres- angiosarcoma) are lacking from such database. In the sion as well as in angiogenesis. current study 25, independent datasets from histologi- Flat port-wine stains are non-tumor malformations of cally different solid tumors were investigated, namely, the skin capillaries [23]. Cutaneous capillary malforma- bladder cancer, glioblastoma, melanoma, breast cancer, tions are usually isolated finding. However, they may lung adenocarcinoma, as indicated in details in Table 1. occasionally coexist with cerebral or ocular vascular mal- Table 2 reports the detailed list of the 90 ion-channels formations, constituting the rare sporadic neurocutane- and ion-channel related genes investigated in the pre- ous Sturge-Weber syndrome (SWS) affecting the cephalic sent study. Briefly, different members were selected from microvasculature, or may represent signs of more aggres- 21 channel families, namely: amiloride-sensitive cation sive vascular malformations or even vascular tumors. channels, Channels voltage-dependent, cation The sympathetic skin response (SSR) is an alteration channels sperm associated, FXYD domain containing ion in skin electrical potential evoked by strong stimuli; it transport regulators, gamma-aminobutyric acid (GABA) consists of a multineuronal reflex activated by a variety receptors, glutamate receptors ionotropic, potassium of afferent inputs where the efferent branches involve channels voltage gated subfamily, cholinergic receptors sympathetic sudomotor fibers. The electrodermal activ- (Nicotinic), chloride channels, cyclic nucleotide gated ity reflects sympathetic cholinergic sudomotor function channels, glutamate Receptors, sodium leak channels, which induces changes in skin resistance to electrical purinergic receptors P2X, sodium-hydrogen exchanger conduction. The response is mediated by ions flux via regulatory factor 4, regulatory solute carrier proteins, activation of receptor-coupled Ca2+, Cl− and K+ chan- sodium channels, activated Ion channels, two nels [24]. Since SSR reflects peripheral C fibers activity, pore segment channels, transient receptor potential cat- it is considered a reliable quantitative measure of sym- ion channels, zinc activated ion channels, . pathetic function and dysfunction as well as in polyneu- Expression of such 90 genes was evaluated in oncomine ropathies and dysautonomic disorders [25–27]. database by setting “cancer vs. normal analysis” and We have previously shown novel serum markers able choosing as cancer type: “superficial bladder cancer” to indicate cardiovascular diseases, [28, 29] as well as within the bladder cancer group, “glioblastoma” within soluble factors able to affect angiogenesis [30] and to dis- the brain and CNS cancer group, “breast invasive criminate infantile hemangioma from more aggressive ductal carcinoma” within the breast cancer group, “lung vascular malformations or tumors [31]. We also investi- adenocarcinoma” within the group, and gated novel molecular markers of melanoma set-up and “melanoma”. progression [32]. Expression fold change (cancer vs. normal samples) and In the present study, we further addressed the issue p values were reported for each analysis. Gene expres- of altered angiogenesis in tumor and non-tumor condi- sion in tissue biopsies from 3673 patients was analyzed. tions. The expression level of 90 ion-channel genes was Namely, 674 control-samples and 2999 cancer-samples investigated in five different solid tumors, having a dif- were investigated. ferent histological origin. The expression level of several Two additional datasets were identified and investi- ion-channels genes was found to be strongly modified; gated within GEO database from NCBI (http://www. Biasiotta et al. J Transl Med (2016) 14:285 Page 3 of 15

Table 1 Tumors types, datasets names, patients numerosity and reference of each dataset investigated in the present study, from oncomine database (http://www.oncomine.org) Tumor type and dataset name No of control samples No of cancer samples References n.

Superficial bladder cancer 1 Dyrskjot dataset 14 28 [86] 2 Lee dataset 68 126 [87] 3 Sanchez dataset 48 28 [88] 4 Blaveri dataset 2 25 [89] Glioblastoma 5 Lee dataset 3 78 [90] 6 Bredel dataset 4 26 [91] 7 Sun dataset 23 81 [92] 8 Murat dataset 4 80 [93] Melanoma 9 Talantov dataset 7 45 [94] 10 Riker dataset 5 14 [95] 11 Critchley dataset 23 23 [96] 12 Haqq dataset 3 23 [97] Breast invasive ductal cancer 13 Ma dataset 28 18 [98] 14 Curtis dataset 144 1556 [99] 15 Radvanji dataset 5 26 [100] 16 Turashvi dataset 20 5 [101] 17 Zhao dataset 4 154 [102] Lung adenocarcinoma 18 Bhattacharjee dataset 17 139 [103] 19 Beer dataset 10 86 [104] 20 Stearman dataset 19 20 [105] 21 Hou dataset 65 45 [106] 22 Okayama dataset 20 226 [107] 23 Selamat dataset 58 58 [108] 24 Landi dataset 49 58 [109] 25 Su dataset 31 31 [110] Totals 674 2999

ncbi.nlm.nih.gov/gds), namely GSE41614 and GSE44115. involving human patients, and to the article n. 16 of the Such datasets specifically refer to the vessels component Oviedo Convention. within cancer samples; they were analyzed by means of For such reasons we submitted to the Ethic Committee the GEO2R interface available at the http://www.ncbi. the request to authorize the in vivo investigation in non- nlm.nih.gov/gds site. tumor patients showing a pathological condition resem- bling, at least in part, the tumor neo-angiogenesis. Such Patients recruitment request was authorized by the Ethic Committee and this Several reports indicate that direct electrical stimulation explains way we investigated the SSR in angioma patients. may affect cell proliferation and dissemination in onco- Fourteen patients (six males and eight females) affected logical setups and may induce other physiological effects by cutaneous flat port-wine stains were recruited at IDI- [33–39]. Thus a clinical-study involving any electrical IRCCS, Rome. The study was approved by the institu- stimulation in cancer patients would be not acceptable by tional review board of IDI-IRCCS Hospital, Rome (IDI the Ethic Committee, according to the articles n. 14 and Ethic Committee 2011, n. 363). Patients with flat port- n. 16 of the Helsinki declaration on biomedical studies wine stains diagnosis (age 18–70 years) undergoing no Biasiotta et al. J Transl Med (2016) 14:285 Page 4 of 15

Table 2 Complete list of ion-channel genes and ion-channel related genes investigated Gene family Gene name Whole gene name

(A) Amiloride-sensitive cation channel 1 ACCN1 Amiloride-sensitive cation channel 1 2 ACCN2 Amiloride-sensitive cation channel 2 3 ACCN3 Amiloride-sensitive cation channel 3 4 ACCN4 Amiloride-sensitive cation channel 4 (B) , voltage-dependent 5 CACNA1A Calcium channel, voltage-dependent, P/Q type, alpha 1A Subunit 6 CACNA1B Calcium channel, voltage-dependent, N type, alpha 1B Subunit 7 CACNA1C Calcium channel, voltage-dependent, L type, alpha 1C Subunit 8 CACNA1D Calcium channel, voltage-dependent, L type, alpha 1D Subunit (C) Cation channel, sperm associated 9 CATSPER1 Cation channel, sperm associated 1 10 CATSPER2 Cation channel, sperm associated 2 11 CATSPER3 Cation channel, sperm associated 3 12 CATSPER4 Cation channel, sperm associated 4 (D) FXYD domain containing ion transport regulator 13 FXYD1 FXYD domain containing ion transport regulator 1 14 FXYD2 FXYD domain containing ion transport regulator 2 15 FXYD3 FXYD domain containing ion transport regulator 3 16 FXYD4 FXYD domain containing ion transport regulator 4 17 FXYD5 FXYD domain containing ion transport regulator 5 (E) Gamma-aminobutyric acid (GABA) A receptor 18 GABRA1 Gamma-aminobutyric acid (GABA) A receptor, alpha 1 19 GABRB3 Gamma-aminobutyric acid (GABA) A receptor, beta 3 20 GABRP Gamma-aminobutyric acid (GABA) A receptor, Pi (F) , ionotropic 21 GRIA1 Glutamate receptor, ionotropic, AMPA 1 22 GRIN2A Glutamate receptor, ionotropic, N-methyl d-aspartate 2A 23 HTR3A 5-Hydroxytryptamine (serotonin) receptor 3A, ionotropic 24 HTR3B 5-Hydroxytryptamine (serotonin) receptor 3B, ionotropic (G) , voltage gated subfamily 25 KCNE3 Potassium channel, voltage gated subfam.E regulatory beta Sub. 3 26 KCNE4 Potassium channel, voltage gated subfam.E regulatory beta Sub. 4 27 KCNH2 Potassium channel, voltage gated Eag related subfamily H, member 2 28 KCNH1 Potassium channel, voltage gated Eag relat.subfam.H, Member 1 29 KCNJ11 Potassium channel, inwardly rectifying subfamily J, member 11 30 KCNJ12 Potassium channel, inwardly rectifying subfamily J, member 12 31 KCNMA1 Potassium channel, calcium activated large conductance subfam.M alpha, member 1 32 KCNMB2 Potassium channel subfamily M regulatory beta subunit 2 33 KCNMB4 Potassium channel subfamily M regulatory beta subunit 4 34 KCNQ1 Potassium channel, voltage gated KQT-Like Subfam. Q, Member 1 35 KCNRG Potassium channel regulator, CLLD4 36 KCNV1 Potassium channel, voltage gated modifier subfamily V, Member 1 37 KCNN1 Potassium channel, calcium activated intermediate/small con- ductance subfamily N alpha, member 1 38 KCNN2 Potassium channel, calcium activated intermediate/small con- ductance subfamily N alpha, member 2 39 KCNN3 Potassium channel, calcium activated intermediate/small con- ductance subfamily N alpha, member 3 Biasiotta et al. J Transl Med (2016) 14:285 Page 5 of 15

Table 2 continued Gene family Gene name Whole gene name

40 KCNN4 Potassium channel, calcium activated intermediate/small con- ductance subfamily N alpha, member 4 (H) Cholinergic receptor, nicotinic 41 CHRNA1 Cholinergic receptor, nicotinic, alpha 1 (muscle) 42 CHRNA2 Cholinergic receptor, nicotinic, alpha 2 (neuronal) 43 CHRNA3 Cholinergic receptor, nicotinic, alpha 3 (neuronal) 44 CHRNA5 Cholinergic receptor, nicotinic, alpha 5 (neuronal) (I) 45 CFTR transmembrane conductance regulator 46 BEST1 47 CLCN1 Chloride channel 1, skeletal muscle (CIC-1) 48 CLCN5 Chloride channel, voltage-sensitive 5 (CIC-5) 49 CLIC1 Chloride intracellular channel 1 50 CLIC4 Chloride intracellular channel 4 (L) Cyclic nucleotide gated channel 51 CNGB3 Cyclic nucleotide gated channel beta 3 (M) Glutamate receptor 52 GRID1 Glutamate receptor, ionotropic, delta 1 53 GRID2 Glutamate receptor, ionotropic, delta 2 (N) Sodium leak channel 54 NALCN Sodium leak channel, non selective (O) P2X 55 P2RX1 Purinergic receptor P2X, ligand gated , 1 56 P2RX2 Purinergic receptor P2X, ligand gated ion channel, 2 57 P2RX3 Purinergic receptor P2X, ligand gated ion channel, 3 58 P2RX4 Purinergic receptor P2X, ligand gated ion channel, 4 59 P2RX6 Purinergic receptor P2X, ligand gated ion channel, 6 60 P2RX7 Purinergic receptor P2X, ligand gated ion channel, 7 (P) Sodium-hydrogen exchanger regulatory factor 4 61 PDZD3 PDZ domain containing 3 (Q) Regulatory solute carrier protein 62 RSC1A1 Regulatory solute carrier protein, family 1, member 1 (R) 63 SCN1A Sodium channel, voltage gated, type I alpha subunit 64 SCN2A Sodium channel, voltage gated, type II alpha subunit 65 SCN3A Sodium channel, voltage gated, type III alpha subunit 66 SCN4A Sodium channel, voltage gated, type IV alpha subunit 67 SCN5A Sodium channel, voltage gated, type V alpha subunit 68 SCN8A Sodium channel, voltage gated, type VIII alpha subunit (S) Glucose activated ion channel 69 SLC5A4 Solute carrier family 5 (glucose activated ion channel) (T) Two pore segment channel 70 TPCN1 Two pore segment channel 1 71 TPCN2 Two pore segment channel 2 (W) Transient receptor potential cation channel 72 TRPC1 Transient recep. potential cation channel, subfamily C, member 1 73 TRPC2 Transient recep. potential cation channel, subfamily C, member 2 74 TRPC3 Transient recep. potential cation channel, subfamily C, member 3 75 TRPC4 Transient recep. potential cation channel, subfamily C, member 4 76 TRPM1 Transient recep. potential cation channel, subfamily M, member 1 Biasiotta et al. J Transl Med (2016) 14:285 Page 6 of 15

Table 2 continued Gene family Gene name Whole gene name 77 TRPM2 Transient recep. potential cation channel, subfamily M, member 2 78 TRPM3 Transient recep. potential cation channel, subfamily M, member 3 79 TRPM4 Transient recep. potential cation channel, subfamily M, member 4 80 TRPM7 Transient recep. potential cation channel, subfamily M, member 7 81 TRPV1 Transient recep. potential cation channel, subfamily V, Member 1 82 TRPV2 Transient recep. potential cation channel, subfamily V, member 2 83 TRPV3 Transient recep. potential cation channel, subfamily V, member 3 84 TRPV4 Transient recep. potential cation channel, subfamily V, member 4 (Y) Zinc activated ion channel 85 ZACN Zinc activated ligand-gated ion channel (Z) Acquaporins 86 AQP1 1 87 AQP2 88 AQP3 89 AQP4 Aquaporin 4 90 AQP5 Aquaporin 5

treatment of any type were consecutively recruited. mean latency and peak-to-peak amplitude were calcu- Patients did not show systemic or neurological disorders lated and used for the following analyses. nor obvious psychological problems. Physical general and neurological examination were normal in all cases. The Statistical analysis average lesion was about 25 cm × 10 cm, typically a por- For gene expression data, the statistically significant tion of the limb. All patients signed an informed consent threshold originally set at p = 0.05 was then corrected to participate in the study. according to the Bonferroni correction for multiple com- parisons, to take into account the multiple comparisons Sympathetic skin responses SSR recording carried out. Therefore, the final corrected threshold was SSR study was carried out according to the techni- set at p < 0.0005 (value obtained from 0.05/90 compari- cal standards of the International Federation of Clinical sons, for each investigated dataset). Neurophysiology [40]. During the test, subjects were kept The expression level of any given gene was considered relaxed with comfortable light and temperature (26– to be significantly altered vs control when the significant 28 °C); the test was started after 5 min of previous adap- p value was matched in at least half—or in at least 1—of tation. The apparatus used was an electromyography and the specific databases investigated for each tumor type. evoked potential equipment (MedelecSynergy, Viasys Namely, regarding the “at least half datasets” stringency Healthcare, Madison WI USA). Recording electrodes level, the significant p value had to be matched in at least consisted of a pair of superficial electrodes: recording 2 datasets out of the 4 investigated for bladder cancer; in was carried out on the glabrous skin on the flat port-wine at least 2 datasets out of the 4 studied for glioblastoma; in stain, and the reference was placed 2 cm away from the at least 2 datasets out of the 4 examined for melanoma; lesion. The ground electrode was proximal to the record- in at least 3 datasets out of the 5 investigated for invasive ing electrodes. Electrical stimulation was applied through ductal breast carcinoma; in at least 4 datasets out of the 8 superficial electrodes over the right median nerve. The investigated for lung adenocarcinoma. stimulus was strong but tolerable (not noxious). The SSR was recorded four consecutive times for each electrical stimulus was applied four times at irregular patient on the diseased skin and four consecutive times intervals of 30–60 s (stimulus duration: 0.1 ms; inten- on the healthy contralateral skin. Mean ± SE was com- sity: 80 mA) to avoid habituation, and SSR waves were puted; mean latency and mean peak-to-peak amplitude obtained. SSR recordings were carried out in quadrupli- recorded at the diseased-skin level were compared to cates at the angiomas lesion sites and onto a contralateral measures obtained on the contralateral healthy control healthy skin region in each patient. site. Paired t Student test was carried out, and statistical The amplifier bandwidth was 0.1–100 Hz. Responses significance was set at p < 0.05. Normal distribution of were recorded on the skin with an impedance <5 kΩ. The SSR data was tested according to the D’Agostino-Pearson Biasiotta et al. J Transl Med (2016) 14:285 Page 7 of 15

test and carried out by the GraphPad software. Data with KCNE4 genes are modified in glioblastoma data- normal distribution were analyzed with the paired t test, sets. KCNE3 shows an average 5.3-fold increase vs. −10 while data with a not-normal distribution were analyzed ctrls (in Lee dataset, p = 3 × 10 and in Sun data- −9 with the non-parametric Wilcoxon match-paired signed set, 5 × 10 ). KCNE4 shows an average 2.9-fold −8 ranked test. increase vs ctrls in Lee dataset (p = 1 × 10 ) and −13 in Sun dataset (p = 1 × 10 ). KCNN4 was found Results altered in nearly all lung carcinoma datasets, namely Expression level of ion‑channels genes in tumors −6 in Bhattacharjee dataset (p = 1 × 10 ), Stearman −7 −9 The expression level of 90 ion-channels and ion-channels dataset (p = 3 × 10 ), Hou dataset (p = 3 × 10 ), −8 related genes (see Table 2 for the complete list) was ana- Okayama dataset (p = 1 × 10 ), Selamat dataset −14 −17 lyzed in 3673 human biopsies of 5 histologically different (p = 2 × 10 ), Landi dataset (p = 2 × 10 ) and −8 solid tumors, namely: superficial bladder cancer, glio- in Su dataset (p = 6 × 10 ) with an average 3.6-fold blastoma, melanoma, invasive ductal breast cancer, lung increase vs ctrls; adenocarcinoma, as reported in Table 1 in more details. •• Within the chloride channel family, expression of Table 3 indicates the ion-channel families showing a CLIC1 gene was found significantly modified in blad- −8 significantly altered expression (p < 0.0005). For each der cancer (in Lee dataset, p = 8 × 10 and Sanchez −5 cancer type, two columns are presented: the left-hand dataset, p = 3 × 10 ) with an average 1.5-fold column (lighten) reports genes indicating a significantly increase vs. ctrls. CLIC1 is also significantly modified −7 modified expression in at least 1—or less than half-of in glioblastoma (in Bredel dataset, p = 3 × 10 and −23 the investigated datasets. The right-hand column of each Sun dataset, p = 2 × 10 ) with an average 5.7-fold tumor type (shadowed) reports in bold all genes show- increase vs. ctrls; ing a significantly modified expression in at least half of •• Within the transient receptor potential cation chan- the examined datasets (see criteria detailed in “Methods” nel family, expression of TRPM3 gene is altered in −5 section). All genes matching the shadowed columns cri- glioblastoma, i.e., in the Lee dataset (p = 5 × 10 ) −5 teria were found overexpressed. More in detail: and in the Sun dataset (p = 2 × 10 ) with an aver- age 2.3-fold increase vs. ctrls. •• Within the calcium channel, voltage-dependent fam- ily, expression of CACNAD1 gene was found signifi- Within the shadowed columns, glioblastoma appears to cantly modified in superficial bladder cancer data- have the highest number of modified ion-channel genes −6 sets, namely in Dyrskjot dataset (p = 4 × 10 ) and (namely: FXYD5, KCNE3, KCNE4, CLIC1, TRPM3). −8 in Sanchez dataset (p = 5 × 10 ), with an average Melanoma and breast cancer show no genes in the 3.7-fold increase vs. ctrls; shadowed columns. However, within the lighten col- •• Within the FXYD domain containing ion transport umns, breast invasive ductal cancer and melanoma show regulator family, expression of FXYD3 gene was significantly modified ion-channel genes from 13 families found significantly modified in bladder cancer (Dys- and 6 families, respectively. −6 −6 kjot dataset, p = 1 × 10 , Lee dataset, p = 3 × 10 −21 SSR recording in flat port‑wine stains patients and Sanchez dataset, p = 6 × 10 ) with an aver- age 3.3-fold increase vs. ctrls. Furthermore, FXYD5 The above reported analyses demonstrated that the gene expression was found significantly modified in expression level of several ion-channels is significantly −5 glioblastoma (in Lee dataset, p = 2 × 10 and in altered in several human cancer biopsies. Such tumors −11 Sun dataset, p = 1 × 10 ) with an average 2.5-fold are histological different. However, they all present an increase vs. ctrls; altered vascular tree, due to the tumor neo-angiogen- •• Within the glutamate receptor, ionotropic family, esis. We hence hypothesized that vascular alterations expression of HTR3A gene was found significantly observed in several different cancer types may harbor, modified in 5 lung carcinoma datasets, namely at least to a certain extent, the observed ion-channels −8 in Beer dataset (p = 3 × 10 ), in Hou dataset expression modifications. According to these findings, −6 −13 (p = 7 × 10 ), in Okayama dataset (p = 2 × 10 ), we hypothesized that measuring ion-channel transport −8 in Selamat dataset (p = 1 × 10 ), and in Landi data- may represent a non-invasive technique to investigate −6 set (p = 8 × 10 ) with an average 5.08 fold increase alterations in tumor- as well non-tumor altered angio- vs ctrls; genesis. In vivo analysis of ion transport in a vascular •• Within the potassium channel, voltage gated fam- malformation, the clinical model was carried out. In ily, expression of three genes was found significantly fact, measuring in vivo ion-transport in tumor patients modified in two cancer types. Namely, KCNE3 and was considered ethically not acceptable; we were then Biasiotta et al. J Transl Med (2016) 14:285 Page 8 of 15

Table 3 Genes reported show a significantly altered expression in each cancer type Bladder cancer Breast cancer Glioblastoma Lung carcinomaMelanoma modif. modif. modif. in modif. in modified in modif. in at modified in in at modified in modified in modif. in in at am. at least at least

nf at least 1 least half at least 1 least at least 1 at least 1 at least 1 least half half dataset datasets dataset half dataset dataset dataset half datasets datasets

Chan datasets datasets ACCN1 A ACCN3 ACCN3 CACNA1A CACNA1A B CACNAD1 CACNAD1 CACNA1D CACNA1B CATSPER1 CATSPER1 C CATSPER2 CATSPER2 CATSPER3 FXYD5 FXYD3 D FXYD3 FXYD5 FXYD5 FXYD5 FXYD3 FXYD5 E GABRB3 GABRB3 GRIN2A GRIA1 F HTR3A HTR3A HTR3A GRIK1 KCNE4 KCNH2 KCNE3 KCNN4 KCNH1 KCNE4 KCNE4 KCNJ13 KCNN1 KCNE3 G KCNJ11 KCNMA1 KCNJ11 KCNN4 KCNQ1 KCNN4 KCNE4 KCNMA1 KCNQ1 KCNQ1 KCNN2 KCNQ1 KCNRG KCNN1 KCNN2 CHRNA1 CHRNA2 CHRNA5 H CHRNA1 CHRNA3 CHRNA5 CLICN5 I CLIC1 CLIC1 CLIC1 CLCN5 CLCN1 CLIC1 L CNGB3 CNGB3 N NALCN P2RX2 P2RX4 O P2RX4 P2RX4 P2RX4 P2RY13 Q RSC1A1 SCN1A SCN4A R SCN2A SCN3A SCN8A T TPCN1 TPCN1 TPCN2 TRPC2 TRPM2 TRPM4 TRPM2 TRPC4 W TRPM4 TRPV1 TRPM3 TRPM3 TRPM4 TRPM1 TRPV4 TRPV2 TRPV1 TRPV4 AQP2 Z AQP3 AQP4 AQP3 AQP3

For each cancer type the lighten column reports ion-channels genes significantly modified in at least 1 dataset. The shadowed column reports ion-channels genes significantly modified in at least half of the investigated datasets. Channel family codes as in Table 2 Significance threshold: p < 0.0005 (see Methods“ ” section) forced to identify a non-tumor clinical condition show- Therefore, 14 patients with flat port-wine stains diagnosis ing clear vascular anomalies. The flat port-wine stains were consecutively recruited, namely eight female (mean clinical model was approved by the Ethic Committee as age 27 years) and six male (mean age 29.1 years). SSR a safe model to investigate Sympathetic Skin Responses recordings were carried out in quadruplicates at the angi- and electrical signal transport, as the less invasive omas lesion sites and onto a contralateral healthy skin approach possible in the current study. SSR depends on region in each patient. Statistical analysis was performed Ca2+, K+, and Cl− channels found to be altered in Table 3. with paired tests as reported in Methods. Biasiotta et al. J Transl Med (2016) 14:285 Page 9 of 15

Figure 1 shows the mean latency and means (two from skeletal muscle normal uninvolved tissue and amplitude measured at diseased and healthy sites, two pooled RNA from normal tissues). expressed in mV. A strong and significant reduc- Tables 4 and 5 report the gene name, the log2 fold tion in both latency and peak-to-peak amplitude sig- change, the p values adjusted according to the Benjamini nals was observed in the diseased site as compared and Hochberg false discovery rate methods, and the rank to the healthy site, in the whole patients population. position (according to the by p value). Several genes, When a gender-specific analysis was carried out, both within the top 250 genes most significantly regulated, latency and amplitude were strongly reduced in female belong to ion channels families, either in bladder cancer patients, while latency was reduced in male, although blood vessels (9 genes, Table 4) and in angiosarcoma ves- in a not-significant manner. sels (123 genes, Table 5). Such data indicate that several ion channels may play a key role in the vessels within Ion channel expression in normal vessels as compared cancer tissues. to tumor derived vessels Data reported in Table 3 and in Fig. 1 led us to hypoth- Discussion esize that ion channels genes may play a relevant role in It is widely known that nerves and vessels follow similar cancers as well as in other pathological conditions of anatomical paths. Often nerves and vessels show an over- altered angiogenesis. To further support this hypothesis lapping anatomy with overlapping branches and ramifi- we investigated two human samples datasets available at cations. Several molecular factors are reported to control GEO database. The first dataset (GSE41614) reports tran- their respective patterns and growth in a coordinated scriptional profiling of tumor-associated blood vessels in manner [41], including semaphorin, netrin and slit [42], human invasive bladder cancer samples. In such dataset all strongly regulated by Ca2+, Na+, and Cl− channels the expression data were obtained on laser capture micro- [43, 44]. Thus, an architectural or functional modifica- dissected vessels isolated from normal bladder tissue or tion of the ones may affect the architecture or function from tumor bladder tissues. Data from ten samples were of the others. We, therefore, argued that unordered angi- analyzed, i.e., five normal samples vs five cancer samples. ogenesis occurring in tumors and vascular malforma- The second dataset (GSE44115) reports gene expres- tions may associate to a corresponding unordered nerve sion data in OCT frozen human angiosarcoma compared formation and therefore to a measurable alteration of to OCT frozen normal mesenchymal tissues. In this case the electric stimulus transport. We hypothesized that (i) 18 angiosarcoma samples were compared to four controls ion channels (which are known to regulate nerve- and

Fig. 1 SSR recordings carried out in 14 flat port-wine stains patients. Both latency and amplitude signals were measured on the angioma site and the contralateral healthy site of each patient. A paired test was carried out according to “Methods” section. **indicates p < 0.01; *indicates p < 0.05 Biasiotta et al. J Transl Med (2016) 14:285 Page 10 of 15

Table 4 Ion channels genes most regulated in bladder cancer vessels vs control vessels Rank position in the top 250 Gene identifier Gene name Adjusted p value* log2 fold change (by p value)

16th KCNC4 Potassium voltage-gated channel subfamily C mem- 0.009 0.507 ber 4 + 42th KCNG4 Potassium voltage-gated channel modifier subfamily 0.01 0.415 G member 4 + 75th VDAC3 Voltage dependent anion channel 3 0.01 1.511 − 90th CRACR2B Calcium release activated channel regulator 2B 0.01 0.472 + 115th KCNS2 Potassium voltage-gated channel modifier subfamily 0.01 0.637 S member 2 + 153th SEC23B Sec23 homolog B, coat complex II component 0.01 0.566 (involved in vesicle trafficking) − 173th CBARP CACN Beta subunit associated regulatory protein 0.01 0.627 + 218th P2RX5 Purinergic receptor P2X 5 (ligand-gated ion channel) 0.01 0.403 + 247th SCN2B Sodium voltage-gated channel beta subunit 2 0.02 0.487 + * According to the Benjamini and Hochberg false discovery rate method Data from Geo dataset GSE41614, available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc GSE41614) =

Table 5 Ion channels genes most regulated in angiosarcoma vs controls Rank position in the top Gene identifier Gene name Adjusted p value* log2 fold change 250 (by value)

3th KCNJ16 Potassium voltage-gated channel subfamily J member 16 0.0000003 6.673 + 41th CLCNKA Chloride voltage-gated channel Ka 0.001 3.516 + 52th HCN2 Hyperpolarization activated cyclic nucleotide gated potas- 0.001 2.099 sium channel 2 + 53th KCNQ2 Potassium voltage-gated channel subfamily Q member 2 0.001 1.276 + 61th FXYD4 FXYD domain containing ion transport regulator 4 0.0009 6.247 + 75th CRACR2B Calcium release activated channel regulator 2B 0.002 1.117 + 104th AQP10 Aquaporin 10 0.004 2.096 + 125th KCNJ15 Potassium voltage-gated channel subfamily J member 15 0.004 3.203 + 147th KCNK12 Potassium two pore domain channel subfamily K member 0.004 2.008 12 + 153th FXYD2 FXYD domain containing ion transport regulator 2 0.005 2.596 + 167th KCNJ1 Potassium voltage-gated channel subfamily J member 1 0.01 3.369 + 250th AQP2 Aquaporin 2 0.01 8.359 + * According to the Benjamini and Hochberg false discovery rate method Data from Geo dataset GSE44115, available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc GSE44115) = vessel- formation) may show altered expression in reported in the shadowed columns of Table 3 selected tumors and (ii) SSR recording in vascular malformations according to highly stringent criteria, i.e. to the ion-chan- patients may unreveal a clinical non-invasive sign of the nel genes found altered in at least half of the investigated unordered vessels formation. databases of each tumor. We report in the current study The expression level of members of several ion-channel that such genes show an increased expression in cancer families was found significantly modified in histologically vs. ctrls samples and have in most cases a definite role different human cancer biopsies, according to the meas- in vascular biology and/or cancer setup/progression or ures reported in Oncomine database, in an almost 4000 nervous system biology. patients-vs-controls group. While several genes were CACNA1D, a calcium related transporter gene, shows found significantly modified (p < 0.0005) in at least one an average 3.75-fold increased expression in superfi- human dataset, we limit here the discussion to the genes cial bladder cancer datasets. No reports are present in Biasiotta et al. J Transl Med (2016) 14:285 Page 11 of 15

literature referring CACNA1D direct link to bladder can- transmission [69] and the T-lymphocytes maturation cer. Nevertheless, some studies relate its expression- and [70] and it is known to regulate K-channels in vascular methylation-level to prostate cancer [45, 46], indicating smooth muscle [71] and more in general neuronal excit- CACNA1D as a possible regulator of prostate cancer ability. Furthermore, KCNN4 single-nucleotide polymor- aggressiveness [47], or refer it to CNS disorders [48], dia- phisms have been related to myocardial infarction [72]. betes [49] or calcium level within the vessels [50]. Most interestingly, KCNN4 has been linked to vascular FXYD3 shows a 3.3 average fold increase expression cells proliferation [73]. Notably, expression of several in bladder cancer. It encodes a protein potassium channels is modified in bladder cancer vessels regulating ion-pumps and ion-channels function and is vs controls (KCNC4, KCNG4, KCNS2, see Table 4) and known to have a role in tumor progression. Its activity in angiosarcoma vs controls (namely KCNJ16, KCNQ2, is related to glucose and Cl− ions and has been indicated KCNJ15, KCNJ12, KCNJ1, see Table 5). as a possible biomarker in bladder cancer [51, 52] as well Within the Cl- intracellular channels, CLIC1 expres- as other cancers including breast [53], colorectal cancer sion was found altered in bladder cancer and glioblas- [54], endometrial cancer [55] and intrahepatic cholangio- toma. CLIC1 has been previously found up-regulated in carcinoma [56]. glioblastoma [74], it is involved in different tumors and FXYD5 is a transmembrane auxiliary subunit of the acts as an oncogene in pancreatic cancer [75], and has Na+-K+-ATPase; it shows a 2.5-fold increase in glioblas- been indicated as a possible cancer biomarker [76]. Inter- toma. No direct link with glioblastoma has been reported estingly, CLIC1 has recently shown a key role in angio- to date, however it has been found up-regulated in ada- genesis control in combination with integrins [77, 78]. mantinomatous craniopharyngiomas in children [57] Interestingly, expression of one Cl− channel (namely and other members of the FXYD family are known to be CLCNKA) is strongly modified in angiosarcoma vs con- associated with different cancer types such as urothelial trols (see Table 5). carcinoma [58], esophageal squamous cell carcinoma Within the transient receptor potential cation chan- [59] and cholangiocarcinoma [60]. FXYD channels are nels, TRPM3 gene expression was found increased in involved in the anti-oxidative stress in vascular smooth glioblastoma by 2.3-fold. Its expression has been previ- muscle, thus controlling the vascular tone [61] and blood ously found increased in glioblastoma [79] and is known pressure [62]. Most interestingly, expression of FXYD2 to exhibit mechanosensitivity contributing to vascular and FXYD4 genes is modified in angiosarcoma vs control and cardiac functions [80]. human samples (Table 5). SSR is under the direct control of Ca2+, K−, Cl− ion The expression of serotonin receptor HTR3A was channels and strictly depends on the sympathetic auton- found increased by fivefold in lung carcinoma; no direct omous nerve function. In the current study, several link has been reported between HTR3A and lung adeno- ion channels related to the Ca2+, K+, and Cl− have rel- carcinoma, yet; however, nucleotide polymorphisms of evant, and significantly increased expression in differ- this gene have been related to opioid- or nausea/vomiting ent human cancers and SSR was found strongly altered signaling pathways in cancer patients [63] and to bowel in human vascular malformations. SSR has been previ- syndrome [64]. HTR3 is the only ligand-gated ion chan- ously indicated as a possible useful diagnostic technique nel among the serotonin receptors, and it has been asso- in CNS pathologies [81, 82], as well as fibromyalgia [83] ciated with neurological disorders such as depression and diabetes [84]. Further, autonomic nerve develop- [65] or [66]. Most interestingly about the ment and function has been recently shown to play a key current study, HTR3A acts as a ligand-gated ion channel role in prostate cancer progression [85]. Thus, given the , and causes fast, depolarizing responses known role of several ion channels in the tumor, nerve, in (http://www.genecards.org/cgi-bin/carddisp. and vascular biology, we hypothesize that the altered pl?gene=HTR3A). It is up-regulated in rosacea, i.e., a SSR observed in vascular skin malformations, and the chronic inflammatory skin disease often showing telangi- observed altered ion-channel gene-expression in several ectasias in the erythematotelangiectatic form (ETR) [67]. tumors may represent phenomena related to the unor- Within the potassium intermediate/small conduct- dered nerve- and vessel formation, common to tumor- ance calcium-activated channels, expression of 3 genes related and non-tumor-related vascular anomalies. (namely KCNE3, KCNE4, KCNN4) has been found SSR recording gave more significant results in female altered in the current study, namely in glioblastoma and while in male the trend was present but with no statisti- lung adenocarcinoma. While no direct evidence relate cal significance. Such difference may be related to a dif- KCNE3 or KCNE4 to glioblastoma, KCNE4 is most ferent transport of electrical stimuli in the female skin, abundantly expressed in brain [68]; it exerts functions which appears evident at both healthy (white boxes such as controlling the neuronal firing rate, the synaptic in Fig. 1) and diseased sites (black boxes in Fig. 1), as Biasiotta et al. J Transl Med (2016) 14:285 Page 12 of 15

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